Enzyme catalysis in the presence of ionic liquids

ABSTRACT

The invention relates to the implementation of enzyme-catalysed reactions in the presence of ionic liquids.

[0001] The invention relates to compositions comprising an enzyme andionic liquids as well as a method for carrying out enzyme-catalysedreactions in the presence of ionic liquids.

[0002] To date, enzymes have become firmly established as biocatalystsfor reactions on the laboratory and industrial scale. Nevertheless,despite all the success with enzymatic reactions, there are stillproblems such as, for example,

[0003] low productivities as a result of too low educt solubilities;

[0004] low yields in equilibrium reactions;

[0005] insufficient selectivity in regio- and stereoselectiveconversions;

[0006] product inhibition;

[0007] the occurrence of side reactions (parallel, consecutivereactions).

[0008] There are known attempts to solve this problem by adding organicsolvents (G. Carrea, S. Riva, Angew, Chem. 2000, 112, 2312; J. M. S.Cabral, M. R. Aires-Barros, H. Pinheiro, D. M. F. Prazeres, J.Biotechnol. 1997, 59, 133; M. N. Gupta, Eur. J. Riochem. 1992, 203, 25),by adding salts (A. M. Blinkorsky, Y. L. Khmelnitzky, J. S. Dordick, J.AM, Chem. Soc. 1999, 116, 2697) or by carrying out the reaction inmicroemulsions (B. Orlich, R. Schomähcker 1999, 65, 357-362). Frequentlyhowever, the improvements achieved thereby are not significant and donot justify the additional expenditure, or the enzyme stabilitydecreases severely under these conditions (G. Carrea, S. Riva, Angew.Chem. 2000, 112, 2312). At low temperatures (<100° C.) ionic liquids aremelting salts which represent a new class of solvents having anon-molecular ionic character. Although the first representatives havebeen known since 1914, ionic liquids have only been investigatedintensively as solvents for chemical conversions in the last 15 years.Ionic liquids have no measurable vapour pressure. This is a majoradvantage from the process engineering point of view because in thisway, the distillative separation of a reaction mixture is possible as aneffective method for product separation. The known problems caused byazeotrope formation between solvents and products do not occur. Ionicliquids are temperature-stable up to above 200° C. By means of asuitable choice of cation and anion, it is possible to gradually adjustthe polarity and thereby tune the solubility properties. The range goesfrom water-miscible ionic liquids through water-immiscible ionic liquidsas far as those which themselves form two phases with organic solvents.The skilful utilization of the extraordinary solubility properties isthe key to the successful use of ionic liquids as a new class ofsolvents.

[0009] Ionic liquids have already been successfully used as new types ofmedia in two-phase catalysis or as the medium for liquid-liquidextraction). (P. Wasserscheid, W. Keim, Angew. Chem. 2000, 112, 3926).

[0010] According to the invention a substantial increase in the yieldand selectivity was surprisingly established during the conversion of awide range of educts with different enzymes in the presence of ionicliquids, which represents a significant improvement compared with theprior art. No adverse effects of the ionic liquid on the enzymestability were established and in individual cases, even a stabilisingeffect was found.

[0011] This is unexpected and surprising bearing in mind th ionic natureof the ionic liquids and the strong interactions thereby possiblebetween the ionic liquids and the enzyme with its likewise chargedgroups.

[0012] It was also found that ionic liquids can be used as co-solventsto improve the solubility of educts and products.

[0013] The invention relates to a method for the conversion ofsubstances (educts) in the presence of enzymes as a catalyst in areaction medium comprising ionic liquids.

[0014] The ionic liquid can be miscible with water or immiscible withwater. In the same way, it is possible to carry out a single-phase,two-phase or multi-phase reaction.

[0015] The ionic liquids comprise compounds having the general formula

[A]_(n) ⁺[Y]^(n−),

[0016] where

[0017] n 1 or 2 and

[0018] the anion [Y]^(n−) is selected from the group comprisingtetrafluoroborate ([BF₄]⁻), tetrachloroborate ([BCl₄]⁻),hexafluorophosphate ([PF₆]⁻), hexafluoroantimonate ([SbF₆]⁻),hexafluoroarsenate ([AsF₆]⁻), tetrachloroaluminate ([AlCl₄]⁻),trichlorozincate [(ZnCl₃]⁻), dichlorocuprate, sulphate ([SO₄]²⁻),carbonate ([CO₃]²⁻), fluorosulphonate, [R′—COO]⁻, [R′—SO₃]⁻ or[(R′—SO₂)₂N]⁻, and R′ is a linear or branched aliphatic or alicyclicalkyl containing 1 to 12 carbon atoms or a C₅-C₁₈-aryl,C₅-C₁₈-aryl-C₁-C₆-alkyl or C₁-C₆-alkyl-C₅-C₁₈-aryl radical that can besubstituted by halogen atoms, the cation [A]⁺ is selected from

[0019] quaternary ammonium cations having the general formula

[NR¹R²R³R]⁺,

[0020] phosphonium cations having the general formula

[PR¹R²R³R]⁺,

[0021] imidazolium cations having the general formula

[0022]  where the imidizole nucleus can be substituted with at least onegroup selected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl,C₅-C₁₂-aryl or C₅-C₁₂-aryl-C₁-C₆-alkyl groups,

[0023] pyridinium cations having the general formula

[0024]  where the pyridine nucleus can be substituted with at least onegroup selected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl,C₅-C₁₂-aryl or C₅-C₁₂-aryl-C₁-C₆-alkyl groups,

[0025] pyrazolium cations having the general formula

[0026]  where the pyrazole nucleus can be substituted with at least onegroup selected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl,C₆-C₁₂-aryl or C₅-C₁₂-aryl-C₁-C₆-alkyl groups,

[0027] and triazolium cations having the general formula

[0028]  where the triazole nucleus can be substituted with at least onegroup selected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl,C₅-C₁₂-aryl or C₅-C₁₂-aryl-C₁-C₆-alkyl groups,

[0029]  and the radicals R¹, R², R³ are selected independently of oneanother from the group consisting of

[0030] hydrogen;

[0031] linear or branched, saturated or unsaturated, aliphatic oralicyclic alkyl groups with 1 to 20 carbon atoms;

[0032] heteroaryl, heteroaryl-C₁-C₆-alkyl groups with 3 to 8 carbonatoms in the heteroaryl radical and at least one heteroatom selectedfrom N, O and S which can be substituted with at least one groupselected from C₁-C₆-alkyl groups and/or halogen atoms;

[0033] aryl-, aryl-C₁-C₆-alkyl groups with 5 to 12 carbon atoms in thearyl radical which if necessary can be substituted with at least oneC₁-C₆-alkyl group and/or one halogen atom.

[0034] In a further aspect the invention relates to a compositioncomprising an enzyme and at least one of the ionic liquids definedabove. These compositions can be used as the starting point for carryingout the afore-mentioned enzymatically catalysed reactions. Accordingly,in addition to the enzyme (biocatalyst), the compositions according tothe invention can also contain the educts (substrate) to be convertedand as the reaction proceeds, naturally also the reaction productsobtainable by the enzymatic reaction.

[0035] A still further aspect is thus the use of ionic liquids,especially the ionic liquids defined above, as the reaction medium or aconstituent of the reaction medium in biocatalysis, i.e., carrying outenzymatically catalysed reactions on substrates.

[0036] In a particular development of the invention the alkyl, aryl,arylalkyl and alkylaryl sulphonate groups (anion [Y]) can be substitutedby halogen atoms, especially fluorine, chlorine or bromine. Especiallypreferred are the perfluorinated alkyl and afore-mentioned arylsulphonates such as trifluoromethane sulphonate (triflate). Asnon-halogenated representatives mention may be made of methanesulphonate, benzene sulphonate and the toluene sulphonate group as wellas other sulphonate leaving groups known in the prior art.

[0037] In a further development of the invention the alkyl, aryl,arylalkyl and alkylaryl carboxylate groups can be substituted by halogenatoms, especially fluorine, chlorine or bromine, Especially preferredare the fluorinated, in particular the perfluorinated alkyl andabove-mentioned aryl carboxylates, such as trifluoromethane carboxylate(trifluoroacetate; CF₃COO⁻). As non-halogenated representatives mentionmay be made of the acetate and benzoate group as well as all othercarboxylate leaving groups known in the prior art.

[0038] In preferred developments of the invention the C₁-C₆-alkyl groupsmentioned in connection with the substituents can be replaced byC₂-C₄-alkyl groups independently of each other. Likewise, theC₁-C₆-alkoxy groups mentioned in connection with the substituents can bereplaced by C₂-C₄-alkoxy groups independently of each other. In afurther alternative of the invention the C₅-C₁₂-aryl groups mentioned inconnection with the substituents can be replaced by C₆-C₁₀-aryl groupsindependently of each other and the C₃-C₈-heteroaryl groups can bereplaced by C₃-C₆-heteroaryl groups independently of one another. Thehalogen atoms with which the alkyl, alkoxy and aryl groups can besubstituted are selected from fluorine, chlorine, bromine and iodine,preferably fluorine, chlorine and bromine.

[0039] In a preferred development the radical R′ is a linear or branchedaliphatic or alicyclic alkyl containing 1 to 8 carbon atoms or aC₆-C₁₀-aryl, C₆-C₁₀-aryl-C₁-C₄-alkyl or C₁-C₄-alkyl-C₆-C₁₀-aryl radicalwhich can be substituted by halogen atoms.

[0040] The cations [A] are, for example, selected from trimethylphenylammonium, methyltrioctyl ammonium, tetrabutyl-phosphonium,3-butyl-1-methyl-imidazolium, 3-ethyl-1-methyl-imidazolium, N-butylpyridinium, N-ethyl pyridinium, diethyl pyrazolium,1-ethyl-3-methylimidazolium, 1-butyl-3-methylimidazolium,1-hexyl-3-methylimidazolium, 1-octyl-3-methylimidazolium,1-decyl-3-methylimidazolium, 1-butyl-4-methylpyridinium,1-butyl-3-methylpyridinium, 1-butyl-2-methylpyridinium,1-butyl-pyridinium, butyl-methylimidazolium, nonyl-methyl-imidazolium,butyl-methylimidazolium, hexyl-methyl-imidazolium,octyl-methylimidazolium, 4-methyl-butyl-pyridinium, triethyl ammonium,triethylmethyl ammonium, butylmethylpyridinium, propyl ammonium,methyl-methyl-imldazolium, ethyl-methyl-imidazolium,butyl-methyl-imidazolium.

[0041] Ionic liquids and their production are known in the prior art.For the synthesis of ionic liquids using hexafluorophosphate,tetrafluoroborate, bis(trifluoromethylsulphonyl)amide, perfluoroalkylsulphonate and perfluoroalkyl carboxylate ions, the corresponding halidesalt [cation]⁺X⁻ is first formed and isolated by reacting an amineNR₁N₂R₃, a phosphane PR¹R²R³, an imidazole derivative having the generalformula R¹R²⁺N═CR³—R⁵—R³C═N⁺R¹R² or a pyridinium derivative having thegeneral formula R¹R²N═CR³R⁴⁺ with an alkyl chloride, alkyl bromide oralkyl iodide (F. H. Hurley, T. P. Wier, Jr., J. Electrochem. Soc. 1951,98, 207-212; J. S. Wilkes, J. A. Levisky, R. A. Wilson, C. L. Hussey,Inorg. Chem. 1982, 21, 1263-1264; A. A. K. Abdul-Sada, P. W. Ambler, P.K. G. Hodgson, K. R. Seddon, N. J. Steward, WO-A-95/21871) R. H. Dubois,M. J. Zaworotko, P. S. White, Inorg. Chem. 1989, 28, 2019-2020; J. F.Knifton, J. Mol. Catal. 1987, 43, 65-78; C. P. M. Lacroix, F. H, M.Dekker, A. G. Talma, J. W. F. Seetz, EP-A-0989134). Starting from the[A]⁺X⁻ halide salt which has been formed and isolated, two differentpaths are known for the synthesis of ionic liquids usinghexafluorophosphate, tetrafluoroborate, bis(trifluoromethylsulphonyl)-amide, perfluoroalkyl sulphonate and perfluoroalkylcarboxylate ions. On the one hand, the halide salt is converted by theaddition of a metal salt MY (with precipitation or separation of thesalt MX or the product [A]⁺[Y]⁻ from the respective solvent used) where[Y]⁻ stands for a hexafluorophosphate, tetrafluoroborate,bis(trifluoromethyl sulphonyl)amide, perfluoroalkyl sulphonate andperfluoroalkyl carboxylate ion and M⁺ stands for an alkali cation (J. S.Wilkes, M. J. Zaworotko, J. Chem. Soc. Chem. Commun. 1992, 965-967; Y.Chauvin, L. Muβmann, H. Olivier, Angew. Chem. 1995, 107, 2941-2943; P.A. Z. Suarez, J. E. L. Dullius, S. Einloft, R. F. de Souza, J. Dupont,Polyhedron, 1996, 15, 1217-1219; P. Bonhôte, A.-P. Dias, N.Papageorgiou, K. Kalyanasundaram, M. Grätzel, Inorg. Chem. 1996, 35,1168-1178; C. M. Gordon, J. D. Holbrey, A. R. Kennedy, K. R. Seddon, J.Mater. Chem. 1998, 8, 2627-2638; P. A. Z. Suarez, S. Einloft, J. E. L.Dullius, R. F. de Souza, J. Dupont, J. Chim. Phys. 1998, 95, 1626-1639;A. J. Carmichael, C. Hardacre, J. D. Holbrey, M. Nieuwenhuyzen, K. R.Seddon, Anal. Chem. 1999, 71, 4572-4574; J. D. Holbrey, K. R. Seddon, J.Chem. Soc., Dalton Trans. 1999, 2133-2140). On the other hand, by theaddition of a strong acid H⁺[Y]⁻ the halide ion is displaced with therelease of H⁺X⁻ and exchanged for [Y]⁻, where [Y]⁻ here stands for ahexafluorophosphate, tetrafluoroborate, bis(trifluoromethylaulphonyl)amide, perfluoroalkyl sulphonate and perfluoroalkyl carboxylate ion (J.Fuller, R. T. Carlin, H. C. de Long, D. Haworth, J. Chem. Soc. Chem.Commun. 1994, 299-300). However, ionic liquids can especiallyadvantageously be produced halide-free using the method described in EP00118441.5.

[0042] In a development of the method according to the invention theionic liquid is used as the only reaction medium, i.e., free fromfurther solvents. The fraction of the ionic liquid in the reactionmedium can however be between 0.1 and 99.9 percent by volume, preferablybetween 5 and 75 percent by volume, more preferably between 15 or 50 and75 percent by volume relative to the total quantity of the reactionmedium.

[0043] In addition to the ionic liquid, the reaction medium can alsocontain a further solvent. This can be selected from the groupconsisting of water, buffer solutions (pH 2 to 10, preferably 5 to 8)and organic solvents. Usable organic solvents are miscible with water orimmiscible with water. AB examples of organic solvents mention may bemade of methyl-tert-butyl ether, toluene, hexane, heptane, tert-butanol,glycols, polyalkylene glycols. In addition, however, fundamentally allconventional solvents known in the field of enzyme catalysis can beconsidered.

[0044] All enzymes in EC classes 1 to 6 can fundamentally be considered.The enzyme classification is recommended by the “Nomenclature Committeeof the International Union of Biochemistry and Molecular Biology”(IUBMB). The enzyme is either homogeneously dissolved but can also beused as a suspension or as an immobilisate on an inert carrier.

[0045] It was found according to the invention that the presence ofionic liquids in the reaction medium during enzymatically catalysedreactions results in an improvement in the substrate solubility(biocompatibility), an improvement in the enzyme activity, animprovement in the selectivity, a reduction in product inhibition,suppression of aide reactions (parallel, consecutive reactions) and/oran increase in enzyme stability. The examples prove that enzymes fromdifferent classes can be used, wherein the use of ionic liquids offerssignificant advantages, such as, for example, an increase in theactivity in the case of formate dehydrogenase, a significant increase inthe yield in reactions with galactosidase, an increase in theenantioselectivity for lipases and an improvement in the eductsolubility for hydrophobic educts.

[0046] According to the invention, the enzyme together with the totalquantity of ionic liquid or a part thereof can be used repeatedly or incontinuously operated reactors.

[0047] The enzymes can be selected from the class of oxidoreductases forregio- and stereoselective oxidation and reduction, from the class ofglycosidases for the synthesis of oligosaccharides, from the class oflipases to obtain optically active products (among others, alcohols,amines, carboxylic acids) and from the class of lyases for synthesis,and hydrolases.

[0048] The method according to the invention can be carried out attemperatures between −10° C. and 130° C., preferably in a temperaturerange between 10° C. and 80° C., especially preferably in a temperaturerange between 20° C. and 40° C.

[0049] The method can be carried out in a single-phase fashion or in amultiphase reaction system.

[0050] Some effects arising from the use of ionic liquids as a reactionmedium or as a constituent of reaction media for enzymatic reactionswill be described below as examples. For example, alcohol dehydrogenasesfrom various sources are used among others for the enzymatic reductionof ketones. The solubility of hydrophobic ketones can be improved by theaddition of organic solvents; however, this generally results in areduction in the enzyme activity and stability (W. Hummel, Biochem. Eng.Biotechnol. 1997, 58, 145; A. Liese, T. Zelinski, M. -R. Kula, H.Kierkels, M. Karutz, U. Kragl, C. Wandrey, J. Mol. Cat. B 1998, 4, 91).In a similar fashion, water-miscible ionic liquids can be added to thereaction medium to increase the educt solubility. For the formatedehydrogenase used for cofactor regeneration, an increase in thereaction rate compared with the purely aqueous system is observed in thesame concentration range of the ionic liquid (see Example 1). Nodeactivation of the enzyme was observed even when the time of influenceof the ionic liquid was fairly long. Thus, ionic liquids offer avaluable possibility for increasing the productivity of enzymaticreactions through an increase in the educt concentration. This isespecially interesting for barely soluble to very barely soluble eductssuch as aromatic ketones or steroids.

[0051] For about 20 years glycosidases have not only been used forbreaking bonds between saccharides but also for the synthesis of di- andoligosaccharides. Despite many attempts to use water-miscible solvents(leads to reduced enzyme stability), by means of the generally expensiveactivation of educts, yields no greater than 31% have been obtained evenin the most recent studies (J. H. Yoon, J. S. Rhee, Carbohydr. Res.2000, 327, 377; M. J. Hernaiz, D. H. G. Crout, J. Mol. Cat. B 2000, 10,403). The main problem in these reactions is the immediately initiatedsecondary hydrolysis of the product, catalysed by the same enzyme.Surprisingly, this secondary hydrolysis is almost completely suppressedin the presence of ionic liquids with otherwise the same enzymeactivity. For the example of β-galactosidase-catalysed synthesis ofN-acetyl lactosamine, an important building block for pharmacologicallyrelevant oligosaccharide, it was shown that the presence of ionicliquids increases the yield above 55% when using lactose as aninexpensive donor. A maximum of 30% is achieved without the addition ofionic liquids; however, the product concentration drops rapidly tovalues of <10% as a result of the secondary hydrolysis. Since thesecondary hydrolysis does not take place in the presence of ionicliquids, a simplified reaction process is obtained since it is notnecessary to follow the reaction and interrupt it at the maximum productyield. The galactosidase is very stable in the presence of ionic liquidsand can be repeatedly used after separation by means of ultrafiltrationwithout any change in the attainable yield and the product formationrate.

[0052] Ionic liquids also offer advantages in reverse hydrolysis for thesynthesis of di- and oligosaccharides. In this case, high eductconcentrations together with additives, usually organic solvents, areused to reduce the water activity. Here ionic liquids especially offerthe advantage of a very good dissolving capacity for carbohydrates. Inthe enzymatic synthesis of lactose, it was possible to increase theyield by a factor of 2 and reduce the reaction time by a factor of 5compared with the literature (K. Ajisaka, H. Fujimoto, H. Nishida,Carbohydr. Res. 180, 35-42 (1988)).

[0053] The use of lipases in the presence of organic solvents in single-or two-phase reactions is prior art (G. Carrea, S. Riva, Angew. Chem.2000, 112, 2312, U. T. Bornscheuer, R. J. Kazlauskas, Hydrolases inOrganic Synthesis—Regio- and stereoselective Biotransformations,Wiley-VCH, Weinheim, 1999, A. Liese, K. Seelbach, C. Wandrey, IndustrialBiotransformations, Wiley-VCH, Weinheim, 2000; M. C. Parker, S. A.Brown, L. Robertson, N. J. Turner, Chem. Commun. 1998, 2247). However,the enzyme has so far conventionally been separated by filtration andthe reaction solution conventionally separated by distillation and thesolvent fed back. The use of ionic liquids permits direct distillativeseparation of the reactands from the reaction mixture even in thepresence of the enzyme so that a simplified procedure results. If thereactands possess suitable volatility, this procedure is not limited tolipases. In an investigation of various lipases for racemate splittingin the presence of ionic liquids it was surprisingly established inseveral cases that the conversion rate and the enantioselectivity are insome cases significantly improved, in individual cases a factor of 5better. The reaction in tert-butylmethyl ether which is also used as asolvent for lipase-catalysed reactions in industrial processes, is usedfor comparison.

[0054] The results show that ionic liquids as a reaction medium forenzymatic conversions have numerous advantages compared with theconditions established as the prior art and can be used as biocompatiblesolvents to specifically influence conversions.

[0055] The invention is described in detail by the following exampleswithout however being restricted to these.

EXAMPLES

[0056] The following abbreviations were used for the description ofcomponents used in the examples: tert-butylmethylether tBME, MTBEButyl-methyl-imidazolium PF₆ ⁻ BMIm⁺ PF₆ ⁻ Nonyl-methyl-imidazolium PF₆⁻ NMIm⁺ PF₆ ⁻ Butyl-methyl-imidazolium BF₄ ⁻ BMIm⁺ BF₄ ⁻Hexyl-methyl-imidazolium BF₄ ⁻ HMIm⁺ BF₄ ⁻ Octyl-methyl-imidazolium BF₄⁻ OMIm⁺ BF₄ ⁻ 4-methyl-butyl-pyridinium BF₄ ⁻ 4-MBPy⁺ BF₄ ⁻Triethylammonium-methyl sulphate Et₃NH⁺ MeSO₄ ⁻Triethylmethylammonium-methyl Et₃NMe⁺ MeSO₄ ⁻ sulphateButylmethyl-pyridinium BF₄ ⁻ BMPy⁺ BF₄ ⁻ Propylammonium-nitrate PrNH₃ ⁺NO₃ ⁻ Methyl-methyl-imidazolium-methyl MeSO₄ ⁻ sulphate MMIm⁺Ethyl-methyl-imidazolium-benzoate EMIm⁺ PhCO₂ ⁻Butyl-methyl-imidazolium-trifluoro BMIm⁺ CF₃SO₃ ⁻ methane sulphonate(=triflate) Butyl-methyl-imidazolium-bis- BMIm⁺ (CF₃SO₂)₂N⁻trifluoromethyl sulphonyl)-imidate

[0057] 1. Formate Dehydrogenase from Candida boidinii (FDH)

[0058] The FDH-catalysed oxidation of formic acid to carbon dioxide bythe reduction of nicotinamide adenine dinucleotide (NAD⁺ to NADH+H⁺) isused as the test reaction to determine the enzyme activity. In theenzyme assay the increase in NADH with time at 25° C. is detectedphotometrically at a wavelength of 340 nm.

[0059] Composition of the enzyme assay: 1 ml buffer solution (50 mMtriethanolamine hydrochloride, 1 mM dithiothreitol, hydrochloric acid)pH 7 is mixed with 0.1 ml of aqueous sodium formate solution (2.4 M) and0.1 ml of enzyme solution (0.7 mg/ml, 8.4 U). The enzyme solutionalready contains the cofactor NAD (6 mM).

[0060] In order to test the influence of water-soluble ionic liquids onthe enzyme activity, the volume of the buffer solution in the assay isgradually reduced by 25 vol % and replaced by the ionic liquid. TABLE 1NAD reduction by formate dehydrogenase from Candida boidinii; enzymeactivity compared with the standard reaction in the buffer solutionIonic liquid 25 vol % 50 vol % 75 vol% MMIm⁺ MeSO₄ ⁻ ± + +

[0061] 2. β-galactosidase from Bacillus circulans for Synthesis ofN-acetyl Lactosamine

[0062] The influence of ionic liquids on the progress of β-Gal-catalysedtranagalactosylation starting from lactose and N-acetyl glucosamine isstudied. For this purpose concentration-time profiles of this synthesisin the presence and absence of ionic liquids are recorded and compared.

[0063] In each series of tests 10 reactions were started in parallel in1 ml GC glasses. At 10 minute intervals the reactions are stopped byboiling at 100° C., the reaction solution is filtered (Minisart RC 4Sartorius injection filter) and the concentration of the reactioncomponents at this time is determined chromatographically (AminexHPX-87H cation exchange column from BioRad with a correspondingpre-column, 0.006 M sulphuric acid as eluent with a flux of 0.8 ml/minand a column temperature of 65° C. Detection is accomplished using UV at208 nm and using the refractive index).

[0064] Composition of the reaction mixture: 0.05 ml buffer solution (65mM KH₂PO₄, 195 mM K₂HPO₄) pH 7,3 is mixed with 0.5 ml N-acetylglucosamine solution (GlcNAc 600 mM or 1.2 M in buffer solution), 0.25ml lactose solution (250 mM in buffer solution) and 0.2 ml enzymesolution (10 mg/ml in buffer solution).

[0065] By exchanging buffer solution for ionic liquid in the substratesolutions, the fraction of ionic liquid in the reaction medium isgradually increased. The following substrate solutions are thusobtained:

[0066] a) 0.50 ml N-acetyl glucosamine solution (600 mM in 1:4 MMIm⁺MeSO₄ ⁻: buffer solution)

[0067] b) 0.50 ml N-acetyl glucosamine solution (600 mM in 1:4 MMIm⁺MeSO₄ ⁻: buffer solution) 0.25 ml lactose solution (250 mM in 1:4 MMIm⁺MeSO₄ ⁻: buffer solution)

[0068] c) 0.50 ml N-acetyl glucosamine solution (1.2 M in 1:2 MMIm⁺MeSO₄ ⁻: buffer solution) 0.25 ml lactose solution (250 mM in 1:2 MMIm⁺MeSO₄ ⁻: buffer solution)

[0069] d) 0.50 ml N-acetyl glucosamine solution (600 mM in 1;4 BMIm⁺H₂PO₄ ⁻/Cl⁻: buffer solution) TABLE 2 Synthesis of N-acetyl lactosamineby β- galactosidase from Bacillus circulans with and without ionicliquid Lactose/ Yield Yield Reaction Fraction GlcNAc [%] [%] aftermedium [vol %] ratio after 60 min 100 min Phosphate 1:2.4  5  3 bufferMMIm⁺ MeSO₄ ⁻ a) 12.5 1:2.4 40 40 b) 18.75 1:2.4 44 43 c) 25 1:4.8 49 55(90 min) BMIm⁺ H₂PO₄ ⁻/ d) 12.5 1:2.4 39 30 Cl⁻

[0070] 3. Enantioselective Acylation of R,S-1-Phenylethanol by Catalysiswith Lipase from Candida antarctica (Type B) in Ionic Liquids

[0071] 4.4 ml of an ionic liquid in accordance with Table 3 ortert-butylmethyl ether are mixed with 122 μl of vinyl acetate and 54 μlof 1-phenyl ethanol so that a substrate solution with approximately 0.1mol/l of 1-phenyl ethanol and 0.3 mol/l of vinyl acetate is obtained.Each 1 mg of lyophilised lipase (>120 U/mg) is mixed with 0.4 ml ofsubstrate solution, thoroughly mixed and incubated at 24° C. for 3-4days, shaking slightly.

[0072] For further processing 100 μl of the reaction formulation ismixed with 1 ml of n-hexane/isopropanol (97.5/2.5 v/v) and mixedthoroughly. This hexane/isopropanol extract is used to determine theconcentrations and enantiomer ratios of 1-phenyl ethanol and1-phenylethyl acetate using HPLC. The conversion and the enantiomerexcess were calculated from these concentrations (see Table 3).

[0073] HPLC Conditions: Column: Guard column Nucleosil C-18 5 μm; 10 mm,4.6 mm ID; Preparatory column Chiracel OJ; 50 mm, 4.6 mm ID; Separatingcolumn Chiracel OJ; 250 mm, 4.6 mm ID Eluent: isocratic; 96.5% (v/v)n-hexane,  3.0% (v/v) isopropanol,  0.5% (v/v) ethanol Flux rate: 1ml/mm Temperature: 38° C. Detection: UV detector (205 nm)

[0074] TABLE 3 Enantioselective acylation of 1-phenyl ethanol, catalysedby lipase from Candida antarctica (Type B): conversion and enantiomerexcess in ionic liquids compared to the standard reaction intert-butylmethyl ether; (+) good to better, (±) the same, (−) poor tonone. Ionic liquid/solvent Conversion Enantiomer excess NMIm⁺ PF₆ ⁻ ± +BMIm⁺ BF₄ ⁻ + + HMIm⁺ BF₄ ⁻ ± + OMIm⁺ BF₄ ⁻ + + 4-MBP⁺ BF₄ ⁻ + + BMIm⁺CF₃SO₃ ⁻ + + BMIm⁺ (CF₃SO₂)₂N⁻ + +

[0075] 4. Enantioselective Acylation of R,S-1-Phenyl Ethanol byCatalysis with Lipase from Candida antarctica (Type A) in Ionic Liquids

[0076] Each 5 mg of lyophilised lipase (>30 U/mg) is mixed with 0.4 mlof a substrate solution as in Example 3. The further procedurecorresponds to that described in Example 3. TABLE 4 Enantioselectiveacylation of 1-phenyl ethanol, catalysed by lipase from Candidaantarctica (Type A): conversion and enantiomer excess in ionic liquidscompared to the standard reaction in tert-butylmethyl ether; Ionicliquid/solvent Conversion Enantiomer excess BMIm⁺ PF₆ ⁻ + + NMIm⁺ PF₆⁻ + + BMIm⁺ BF₄ ⁻ ± + HMIm⁺ BF₄ ⁻ + + OMIm⁺ BF₄ ⁻ + ± BMIm⁺ CF₃SO₃ ⁻ + +

[0077] 5. Enantioselective Acylation of R,S-1-Phenyl Ethanol byCatalysis of Lipase from Pseudomonas _sp. in Ionic Liquids

[0078] Each 3 mg of lyophilised lipase (400 U/mg) is mixed with 0.4 mlof a substrate solution as in Example 3. The further procedurecorresponds to that described in Example 3. TABLE 5 Enantioselectiveacylation of 1-phenyl ethanol, catalysed by lipase from Pseudomonas sp.:conversion and enantiomer excess in ionic 1iquids compared to thestandard reaction in tert-butylmethyl ether; Ionic liquid/solventConversion Enantiomer excess MIm⁺ PF₆ ⁻ ± + 4-MBP⁺ BF₄ ⁻ ± + BMIm⁺CF₃SO₃ ⁻ + + BMIm⁺ (CF₃SO₂)₂N⁻ + +

[0079] 6. Enantioselective Acylation of R,S-1-Phenyl Ethanol byCatalysis of Lipase from Alcaligines sp. in Ionic Solvents

[0080] Each 5 mg of lyophilised lipase (>20 U/mg) is mixed with 0.4 mlof a substrate solution as in Example 3. The further procedurecorresponds to that described in Example 3. TABLE 6 Enantioselectiveacylation of 1-phenyl ethanol, catalysed by lipase from Alcaligines sp.:conversion and enantiomer excess in ionic liquids compared to thestandard reaction in tert-butylmethyl ether, Ionic liquid/solventConversion Enantiomer excess BMIm⁺ PF₆ ⁻ ± + BMIm⁺ BF₄ ⁻ ± + HMIm⁺ BF₄ ⁻± + OMIm⁺ BF₄ ⁻ ± + 4-MBP⁺ BF₄ ⁻ ± + BMIm⁺ CF₃SO₃ ⁻ ± +

[0081] 7. Recycling of the Lipase from Candida antarctica (Type B) inIonic Liquids by Distillation

[0082] 600 mg of lyophilised lipase (approx. 10 U/mg) is mixed with 4 mlof ionic liquid (BMIm⁺ (CF₃SO₂)₂N⁻), 1.2 ml of vinyl acetate and 0.7 ml1-phenyl ethanol and thoroughly mixed. The reaction mixture is incubatedfor 40 min at 40° C.

[0083] The non-converted educts and the reaction product 1-phenylacetate is then distilled off (85° C., 0.06 mbar).

[0084] The enzyme/ionic liquid mixture is cooled, re-mixed with 1.2 mlof vinyl acetate and 0.7 ml of 1-phenyl ethanol and again incubated for40 min at 40° C.

[0085] The reaction sequence involving incubation and distilling off canbe repeated many times without the lipase activity diminishing.

[0086] 8. Synthesis of Lactose by Reverse Hydrolysis withβ-galactosidase from Bacillus circulans

[0087] 100 mmol/l of glucose, 20 mmol/l of galactose and 2 mg/ml ofgalactosidase are incubated at 35° C. in a mixture of water and MMImMeSO₄ for 24 h. The reaction is stopped by boiling for 10 minutes at100° C., the reaction solution is filtered (Minisart RC 4 Sartoriusinjection filter) and the concentration of the reaction components atthis time is determined chromatogaphically (Aminex HPX-87H cationexchange column from BioRad with corresponding preparatory column, 0.006M sulphuric acid as eluent with a flux of 0.8 ml/min and a columntemperature of 65° C. Detection is accomplished using UV at 208 nm andusing the refractive index).

[0088] The fraction of ionic liquid is increased from 0 to 100 per centby volume. As a result of water traces in the ionic liquid and theeducts, a water content of 0.6% is obtained for 100% ionic liquid. After24 hours, the conversion no longer increases. The following lactoseyields are obtained depending on the quantity of ionic liquid: Practionof ionic liquid in % Yield [%] 0 0 10 0 20 0 30 4 40 12 50 15 60 15 7016 80 16 90 16 100 17

1. A method for the conversion of substances in the presence of enzymesas a catalyst in a reaction medium comprising at least one ionic liquid,wherein the enzyme is selected from the group of oxidoreductases,lipases, galactosidases, glycosidases, lyases and enzymes in EC class 6.2. The method according to claim 1, characterized in that the ionicliquid has the general formula [Al_(n) ⁺[Y]^(n−), where n=1 or 2 and theanion [Y]^(n−) 0 is selected from the group comprising tetrafluoroborate([BF₄]), tetrachloroborate ([BCl₄]⁻), hexafluorophosphate ([PF₆]⁻),hexafluoroantimonate ([SbF₆]⁻), hexafluoroarsenate ([AsF₆]⁻),tetrachloroaluminate ([AlCl₄]⁻), trichlorozincate (ZnCl₃]⁻),dichlorocuprate, sulphate ([SO₄]²⁻), carbonate ([CO₃]²⁻),fluorosulphonate, [R′—COO]⁻, [R′—SO₃]⁻ or [(R′—SO₂)₂N]⁻, and R′ is alinear or branched aliphatic or alicyclic alkyl containing 1 to 12carbon atoms or a C₅-C₁₈-aryl, C₅-C₁₈-aryl-C₁-C₆-alkyl orC₁-C₆-alkyl-C₅-C₁₈-aryl radical that can be substituted by halogenatoms, the cation [A]⁺ is selected from quaternary ammonium cationshaving the general formula [NR¹R²R³R]⁺, phosphonium cations having thegeneral formula [PR¹R²R³R]⁺, imidazolium cations having the generalformula

 where the imidizole nucleus can be substituted with at least one groupselected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl, C₅-C₁₂-arylor C₅-C₁₂-aryl-C₁-C₆-alkyl groups, pyridinium cations having the generalformula

 where the pyridine nucleus can be substituted with at least one groupselected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl, C₅-C₁₂-arylor C₅-C₁₂-aryl-C₁-C₆-alkyl groups, pyrazolium cations having the generalformula

 where the pyrazole nucleus can be substituted with at least one groupselected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl, C₅-C₁₂-arylor C₅-C₁₂-aryl-C₁-C₆-alkyl groups, and triazolium cations having thegeneral formula

 where the triazole nucleus can be substituted with at least one groupselected from C₁-C₆-alkyl, C₁-C₆-alkoxy, C₁-C₆-aminoalkyl, C₅-C₁₂-arylor C₅-C₁₂-aryl-C₁-C₆-alkyl groups,  and the radicals R¹, R², R³ areselected independently of one another from the group consisting ofhydrogen; linear or branched, saturated or unsaturated, aliphatic oralicyclic alkyl groups with 1 to 20 carbon atoms; heteroaryl,heteroaryl-C₁-C₆-alkyl groups with 3 to 8 carbon atoms in the heteroarylradical and at least one heteroatom selected from N, O and S which canbe substituted with at least one group selected from C₁-C₆-alkyl groupsand/or halogen atoms; aryl-, aryl-C₁-C₆-alkyl groups with 5 to 12 carbonatoms in the aryl radical which if necessary can be substituted with atleast one C₁-C₆-alkyl group and/or one halogen atom.
 3. The methodaccording to any one of the preceding claims, characterised in that thefraction of the ionic liquid in the reaction medium is 0.1 to 99.9percent by volume.
 4. The method according to any one of the precedingclaims, characterised in that in addition to the ionic liquid, thereaction medium also contains a further solvent.
 5. The method accordingto any one of the preceding claims, characterized in that the furthersolvent is water or an organic solvent.
 6. The method according to anyone of the preceding claims, characterised in that the reaction iscarried out at temperatures of −10° C. to 130° C.
 7. The methodaccording to any one of the preceding claims, characterised in that thereaction is carried out in a single-phase fashion or in a multiphasereaction system.
 8. A composition comprising an enzyme selected from thegroups of oxidoreductases, lipases, galactosidases, glycosidases, lyasesand enzymes in EC class 6 and at least one ionic liquid.
 9. Thecomposition according to claim a, characterised in that the ionic liquidis defined as in claim
 2. 10. The composition according to claim 8 orclaim 9, characterised in that it additionally contains a substrate. 11.The composition according to any one of claims 8 to 10, characterised inthat it contains the ionic liquid as a reaction medium or as aconstituent of the reaction medium.
 12. Use of ionic liquids as areaction medium or as a constituent of the reaction medium forenzymatically catalysed reactions, wherein the enzyme is selected fromthe group of oxidoreductaaes, lipases, galactosidases, glycosidases,lyases and enzymes in EC class
 6. 13. The use according to claim 12 as areaction medium or as a constituent of the reaction medium in reversehydrolysis for the synthesis of di- and oligosaccharides.